Melatonin inhibits cytosolic mitochondrial DNA-induced neuroinflammatory signaling in accelerated aging and neurodegeneration.

Chronic inflammation is a pathologic feature of neurodegeneration and aging; however, the mechanism regulating this process is not understood. Melatonin, an endogenous free radical scavenger synthesized by neuronal mitochondria, decreases with aging and neurodegeneration. We proposed that insufficient melatonin levels impair mitochondrial homeostasis, resulting in mitochondrial DNA (mtDNA) release and activation of cytosolic DNA-mediated inflammatory response in neurons. We found increased mitochondrial oxidative stress and decreased mitochondrial membrane potential, with higher mtDNA release in brain and primary cerebro-cortical neurons of melatonin-deficient aralkylamine N-acetyltransferase (AANAT) knockout mice. Cytosolic mtDNA activated the cGAS/STING/IRF3 pathway, stimulating inflammatory cytokine generation. We found that Huntington's disease mice had increased mtDNA release, cGAS activation, and inflammation, all inhibited by exogenous melatonin. Thus, we demonstrated that cytosolic mtDNA activated the inflammatory response in aging and neurodegeneration, a process modulated by melatonin. Furthermore, our data suggest that AANAT knockout mice are a model of accelerated aging.

Glibenclamide Produces Region-Dependent Effects on Cerebral Edema in a Combined Injury Model of Traumatic Brain Injury and Hemorrhagic Shock in Mice.

Cerebral edema is critical to morbidity/mortality in traumatic brain injury (TBI) and is worsened by hypotension. Glibenclamide may reduce cerebral edema by inhibiting sulfonylurea receptor-1 (Sur1); its effect on diffuse cerebral edema exacerbated by hypotension/resuscitation is unknown. We aimed to determine if glibenclamide improves pericontusional and/or diffuse edema in controlled cortical impact (CCI) (5m/sec, 1 mm depth) plus hemorrhagic shock (HS) (35 min), and compare its effects in CCI alone. C57BL/6 mice were divided into five groups (n = 10/group): naïve, CCI+vehicle, CCI+glibenclamide, CCI+HS+vehicle, and CCI+HS+glibenclamide. Intravenous glibenclamide (10 min post-injury) was followed by a subcutaneous infusion for 24 h. Brain edema in injured and contralateral hemispheres was subsequently quantified (wet-dry weight). This protocol brain water (BW) = 80.4% vehicle vs. 78.3% naïve, p < 0.01) but was not reduced by glibenclamide (I%BW = 80.4%). Ipsilateral edema also developed in CCI alone (I%BW = 80.2% vehicle vs. 78.3% naïve, p < 0.01); again unaffected by glibenclamide (I%BW = 80.5%). Contralateral (C) %BW in CCI+HS was increased in vehicle (78.6%) versus naive (78.3%, p = 0.02) but unchanged in CCI (78.3%). At 24 h, glibenclamide treatment in CCI+HS eliminated contralateral cerebral edema (C%BW = 78.3%) with no difference versus naïve. By 72 h, contralateral cerebral edema had resolved (C%BW = 78.5 ± 0.09% vehicle vs. 78.3 ± 0.05% naïve). Glibenclamide decreased 24 h contralateral cerebral edema in CCI+HS. This beneficial effect merits additional exploration in the important setting of TBI with polytrauma, shock, and resuscitation. Contralateral edema did not develop in CCI alone. Surprisingly, 24 h of glibenclamide treatment failed to decrease ipsilateral edema in either model. Interspecies dosing differences versus prior studies may play an important role in these findings. Mechanisms underlying brain edema may differ regionally, with pericontusional/osmolar swelling refractory to glibenclamide but diffuse edema (via Sur1) from combined injury and/or resuscitation responsive to this therapy. TBI phenotype may mandate precision medicine approaches to treat brain edema.

Adenosine Administration With a Stopcock Technique Delivers Lower-Than-Intended Drug Doses.

STUDY OBJECTIVE: Adenosine administration with a stopcock is the recommended treatment for pediatric patients with acute supraventricular tachycardia. Recent reports suggest that many infants do not respond to the first dose of adenosine administered. Our aim is to determine whether administration of adenosine with a stopcock delivers lower-than-expected drug doses in patients weighing less than 10 kg, corresponding to weights of infants. METHODS: We developed an in vitro model of adenosine delivery. Doses of adenosine corresponding to weights 2 to 25 kg were calculated, using a dose of 0.1 mg/kg, and administered through one port of a stopcock. Distilled water was administered through the second port. The adenosine concentration of the output was measured with mass spectrometry and results were confirmed with spectrophotometry of Evans blue. RESULTS: The mean doses of adenosine delivered through the stopcock increased as weight increased. The mean dose of adenosine delivered was 0.08 mg/kg for weights 2 to 9 kg and 0.1 mg/kg for weights 10 to 25 kg (95% confidence interval for difference of means -0.03 to -0.009). The median dose of adenosine delivered was 0.07 mg/kg (interquartile range [IQR] 0.06 to 0.07 mg/kg), 0.09 mg/kg (IQR 0.08 to 0.09 mg/kg), and 0.1 mg/kg (IQR 0.09 to 0.1 mg/kg) for weights 2 to 5, 6 to 9, and 10 to 25 kg, respectively (rank difference=100; P<.05 for 2 to 5 kg versus 10 to 25 kg). Similar results were obtained with spectrophotometry. CONCLUSION: Administration of adenosine through a stopcock delivers doses lower than intended in patients weighing less than 10 kg, which may account for the decreased response of infants to the first dose of adenosine.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and ß-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

Cerebrospinal fluid baclofen concentrations in patients undergoing continuous intrathecal baclofen therapy.

The aim of this study was to report concentrations of cerebrospinal fluid (CSF) baclofen in children undergoing chronic intrathecal baclofen (ITB) infusions. CSF baclofen concentrations were analyzed in 53 specimens obtained by intrathecal catheter aspiration from 43 participants (28 males, 15 females; range 3-44y, mean 16y [SD 8y 11mo]), with functioning baclofen pumps and catheters. Daily ITB doses ranged from 70 to 1395 microg per day (mean 607 microg per day [SD 363], median 575). Baclofen concentration was quantified by high-pressure liquid chromatography and confirmed by injection onto a gas chromatograph. CSF baclofen concentrations from children receiving either simple continuous or complex infusions ranged from 0.2 to 20.0 microg/ml (mean 4.64 microg/ml, median 3.3 microg/ml). CSF baclofen concentrations from children receiving simple continuous infusions ranged from 0.5 to 12.9 (mean 4.7 microg/ml, median 3.55 microg/ml). There was no correlation between ITB dosage and CSF baclofen concentration. We conclude that baclofen concentration can be measured to determine if baclofen is present in CSF. However, there appears to be no correlation between the ITB dose infused and the corresponding CSF baclofen level.